Gene useful for diagnosis and treatment of aplasia of corpus callosum and aspermatogenesis and use thereof

ABSTRACT

Aplasia of the corpus callosum or aspermatogenesis is diagnosed by investigating existence or expression of the BT-IgSF gene with use of a primer for PCR or probe for hybridization comprising a DNA coding for a novel cell adhesion molecule (BT-IgSF) defined in the following (A) or (B) or a partial sequence thereof:
         (A) a protein comprising the amino acid sequence of the amino acid numbers 1 to 409 of SEQ ID NO: 2;   (B) a protein comprising an amino acid sequence of the amino acid numbers 1 to 409 of SEQ ID NO: 2 including substitution, deletion, insertion or addition of one or several amino acids and having a function as a cell adhesion molecule.

RELATED APPLICATIONS

This application claims priority of Japanese Patent Application No.2001-387853, filed Dec. 20, 2001 and Japanese Patent Application No.2002-209457, filed Jul. 18, 2002. Each of the above applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a novel protein estimated to be a celladhesion molecule involved in development of the corpus callosum andspermatogenesis and a gene coding for the same as well as use of theprotein and the gene. The protein and the gene of the present inventionare useful in pharmaceutical and diagnostic fields.

DESCRIPTION OF THE RELATED ART

It is well known that, upon development of various organs of organisms,protein molecules on surfaces of cells constituting organs (celladhesion molecules) play an important role. It has been found that thesecell adhesion molecules are closely associated with important reactionsin organisms such as recognition of immune cells, inflammation andmetastasis of cancer.

The cell adhesion molecules are primarily classified into integrin,cadherin, selectin, immunoglobulin superfamily and CD44 family dependingon their structures. Of these, the immunoglobulin superfamily is a groupof molecules that have a structure similar to immunoglobulin, and anenormous number of proteins belong to this family and exert a widevariety of functions. For example, molecules that carry out signaltransduction associated with cell adhesion are known. There are alsoprotein molecules known as cytokine receptors and protein moleculesknown as virus receptors. Some are also known as protein moleculesexisting on cell surfaces that regulate cell functions, typicallyimmunological functions. Further, many proteins belonging to theimmunoglobulin superfamily are expected to play important roles inmorphogenesis and development of organs in organisms as cell adhesionmolecules. However, tissues or cells constituting a tissue in which eachprotein is expressed significantly differ depending on individualproteins and cannot be easily inferred by analysis of primary sequence(amino acid sequence) of the proteins.

Meanwhile, there are known cases presenting predominant symptoms offacial malformation, mental retardation, speech disorder and so forthdue to aplasia of the corpus callosum (Genuardi, M., Calvieri, F.,Tozzi, C., Coslovi, R., Neri, G., A new case of interstitial deletion ofchromosome 3q, del(3q)(q13.12q21.3), with aplasia of the corpuscallosum, Clinical Dysmorphology, 3, 292–296, 1994; Ogilvie, C. M.,Rooney, S. C., Hodgson, S. V., Berry, A. C., Deletion of chromosome 3qproximal region gives to a variable phenotype, Clinical Genetics, 53,220–222, 1998), and these cases are considered to be caused by deletionof the chromosome 3. However, its causative gene has not beenidentified, and hence no treatment method has been established.

SUMMARY OF THE INVENTION

An object of the present invention is to isolate a gene coding for anovel cell adhesion molecule and provide a technique concerning theutilization of this gene.

The inventors of the present invention searched a known database for agene having homology to the amino acid sequence of the extracellularregion of macrophage colony stimulating factor (M-CSF) receptor andisolated a cDNA of the gene having the found sequence. As a result ofanalysis of expression of the gene, they found that this gene was highlyexpressed in the testis and brain, in particular, in the corpuscallosum, and accomplished the present invention.

That is, the present invention provides the followings.

-   (1) A DNA which codes for a protein defined in the following (A) or    (B):

(A) a protein comprising the amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2;

(B) a protein comprising an amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2 including substitution, deletion,insertion or addition of one or several amino acids and having afunction as a cell adhesion molecule.

-   (2) The DNA according to (1), which codes for a protein comprising    the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence of    SEQ ID NO: 2 including substitution, deletion, insertion or addition    of one or several amino acids.-   (3) The DNA according to (1), which is a DNA defined in the    following (a) and (b):

(a) a DNA comprising the nucleotide sequence of the nucleotide numbers67–1296 of SEQ ID NO: 1;

(b) a DNA which is hybridizable with the nucleotide sequence of thenucleotide numbers 67 to 1296 of SEQ ID NO: 1 under the stringentconditions and codes for a protein functioning as a cell adhesionmolecule.

-   (4) A DNA which comprises the nucleotide sequence of SEQ ID NO: 1 or    a partial sequence thereof and which is used as a primer for PCR or    a probe for hybridization for investigating existence or expression    of a gene corresponding to the nucleotide sequence of SEQ ID NO: 1.-   (5) The DNA according to (4), which is used for diagnosis of aplasia    of the corpus callosum.-   (6) The DNA according to (4), which is used for diagnosis of    aspermatogenesis.-   (7) The DNA according to any one of (4) to (6), which comprises a    set of a DNA having the nucleotide sequence of SEQ ID NO: 8 and a    DNA having the nucleotide sequence of SEQ ID NO: 9.-   (8) A medicament for gene therapy which comprises a DNA coding for    the amino acid sequence of SEQ ID NO: 1 and which is used for    treatment of aplasia of the corpus callosum.-   (9) A medicament for gene therapy which comprises a DNA coding for    the amino acid sequence of SEQ ID NO: 1 and which is used for    treatment of aspermatogenesis.-   (10) A protein defined in the following (A) or (B):

(A) a protein comprising the amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2;

(B) a protein comprising an amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2 including substitution, deletion,insertion or addition of one or several amino acids and having afunction as a cell adhesion molecule.

-   (11) A medicament comprising the protein according to (10) or a salt    thereof.-   (12) The medicament according to (11), which is used for treatment    of aplasia of the corpus callosum.-   (13) The medicament according to (11), which is used for treatment    of aspermatogenesis.

The protein of the present invention was identified as a proteinmolecule belonging to the immunoglobulin superfamily based on itsstructure, and designated as “BT-IgSF (brain and testis-specificimmunoglobulin superfamily)” protein.

The present invention provides a novel cell adhesion molecule, a BT-IgSFprotein, and a DNA coding for the protein. The BT-IgSF protein and a DNAcoding for the protein are useful for treatment and diagnosis of aplasiaof the corpus callosum or aspermatogenesis.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 (photograph) shows results of Northern hybridization using a cDNAfragment of the human BT-IgSF gene as a probe.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereafter, the present invention will be explained in more detail.

The DNA of the present invention is a DNA coding for a protein definedin the following (A) or (B):

(A) a protein comprising the amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2;

(B) a protein comprising an amino acid sequence of the amino acidnumbers 1 to 409 of SEQ ID NO: 2 including substitution, deletion,insertion or addition of one or several amino acids and having afunction as a cell adhesion molecule.

The DNA of the present invention can be obtained by amplifying mRNAderived from the human kidney by polymerase chain reaction (PCR, White,T. J. et al., Trends Genet., 5, 185, 1989) as described later. Further,since the nucleotide sequence of cDNA of the BT-IgSF gene has beenelucidated by the present invention, the DNA fragment of the presentinvention can also be obtained by chemical synthesis. Further, a genomicgene for the BT-IgSF protein can be obtained by performing PCR usinghuman chromosomal DNA as a template. The BT-IgSF gene derived fromchromosome is expected to include one or more introns in the codingregion, but even such a DNA interrupted with intron(s) also falls withinthe scope of the DNA of the present invention so long as it codes forthe BT-IgSF protein.

As a raw material for obtaining cDNA coding for the BT-IgSF protein,although any of human organs or established cell lines producing theBT-IgSF protein can be utilized, specifically, the kidney can bementioned. Further, cDNA derived from the testis of an animal such asmouse can also be used as a raw material for obtaining a homologue ofthe BT-IgSF gene. Messenger RNA (mRNA) is prepared from cells of tissuesof such organs in a conventional manner (Sambrook, J. et al., MolecularCloning, Vol. 3, Ed. 2, Cold Spring Harbor Laboratory Press, 1989).Further, mRNA derived from the human kidney is commercially available(Clontech, #6538-1) and can be preferably used in the present invention.A single-stranded cDNA is prepared by using the obtained mRNA as atemplate and a reverse transcriptase.

Various oligonucleotides are chemically synthesized as 5′ end primersand 3′ end primers for the BT-IgSF gene, then these primers, thesingle-stranded cDNA and Taq DNA polymerase are used to amplify the DNAfragment by PCR, and a DNA fragment having a target size is prepared byagarose gel electrophoresis. The nucleotide sequence of the prepared DNAfragment can be directly determined. Examples of the primers used forPCR include oligonucleotides having the nucleotide sequences of SEQ IDNOS: 8 and 9. If PCR is performed by using these primers and mRNA orcDNA library derived from a human tissue as a template, there can beobtained a DNA having one nucleotide of G at the 5′ end and a sequenceof GACATGAGG at the 3′ end in addition to the sequence of SEQ ID NO: 1.

Further, the nucleotide sequence can also be determined by inserting anamplified fragment into an appropriate commercially available plasmidvector such as pCR2.1, pCRII (both from Invitrogen) or pUC118 (TakaraShuzo), transforming Escherichia coli such as INValphaF′ (Invitrogen) orHB101 (Takara Shuzo) and purifying the plasmid.

The result of determination of the nucleotide sequence of DNA of thepresent invention in a conventional manner is shown in SEQ ID NO: 1, andthe amino acid sequence encoded by this DNA is shown in SEQ ID NO: 2.

As a result of search of the amino acid sequence of SEQ ID NO: 2 usingSimple Modular Architecture Research Tool (SMART, see Schulta, J.,Copley, R. R., Doerks, T., Ponting, C. P., Bork, P., SMART: A web-basedtool for the study of genetically mobile domains, Nucleic AcidsResearch, 28 (1), 231–234, 2000; http://smart.embl-heidelberg.de), thissequence was found to be constituted by a signal peptide, extracellularregion, transmembrane region and intracellular region from theN-terminus side. Of these, the signal sequence was expected to comprisethe residues of the amino acid numbers −22 to −1 of SEQ ID NO: 2.Therefore, the matured protein of BT-IgSF is expected to correspond tothe amino acid numbers 1 to 409 of SEQ ID NO: 2.

On the other hand, in an analysis using the PSORT (Nakai, K.,“Prediction of structure and function of proteins”, Idensi Igaku(Genetic Medicine), Vol. 4, 3, 377–382, 2000; http://psort.nibb.ac.jp)as an analysis program, the signal sequence was expected to comprise theresidues of the amino acid numbers −22 to −2 of SEQ ID NO: 2.

Based on the above, the matured protein of BT-IgSF is expected tocorrespond to the residues of the amino acid numbers 1 to 409 or theamino acid numbers −1 to 409 of SEQ ID NO: 2. In either case, thematured protein of BT-IgSF includes the residues of the amino acidnumbers 1 to 409.

In addition, the transmembrane region was expected to comprise theresidues of the amino acid numbers 224 to 246 in the analysis by SMARTand the residues of the amino acid numbers 228 to 244 in the analysis byPSORT.

The DNA of the present invention is a DNA fragment coding for a BT-IgSFprecursor having the amino acid sequence of SEQ ID NO: 2 (amino acidnumbers −22 to 409) or an amino acid sequence of the amino acid numbers1 to 409 or the amino acid numbers −1 to 409 of SEQ ID NO: 2. In thepresent invention, the matured BT-IgSF protein may be added with amethionine residue at the N-terminus of the amino acid sequence of theamino acid numbers 1 to 409 or the amino acid numbers −1 to 409 of SEQID NO: 2.

In the present invention, the BT-IgSF protein may include substitution,deletion, insertion or addition of one or more amino acid residues solong as the activity as the BT-IgSF protein, that is, a function as acell adhesion molecule, is not substantially deteriorated. A DNA codingfor any of these BT-IgSF proteins also falls within the scope of thepresent invention. Specific examples of such a DNA fragment include aDNA fragment comprising the nucleotide sequence of the nucleotidenumbers 1 to 1296 of SEQ ID NO: 1 and a DNA fragment comprising thenucleotide sequence of the nucleotide numbers 67 to 1296 of SEQ IDNO: 1. Further, sequences in which a codon for each amino acid isreplaced with an equivalent codon are also encompassed in the scope ofthe present invention so long as the nucleotide sequences code for thesame amino acid sequence.

A DNA coding for a protein substantially the same as such BT-IgSFproteins as mentioned above can be obtained by, for example, thesite-directed mutagenesis to modify the nucleotide sequence so thatamino acid residues at a specific site should include substitution,deletion, insertion, addition or inversion. Further, such a DNA can alsobe obtained by treating a DNA coding for a BT-IgSF protein by amutagenizing agent or the like to randomly introduce a mutation. A DNAcoding for a protein substantially the same as the BT-IgSF proteins canbe obtained by expressing such a DNA introduced with a mutation in anappropriate cell and investigating activity of the expression product asa cell adhesion molecule. Further, a DNA coding for a proteinsubstantially the same as the BT-IgSF proteins can also be obtained byisolating a DNA that is hybridizable with DNA having, for example, thenucleotide sequence of the nucleotide numbers 1 to 1296 or thenucleotide sequence of the nucleotide numbers 67 to 1296 of SEQ ID NO: 1in Sequence Listing under the stringent conditions, and codes for aprotein having a function as a cell adhesion molecule from a cellharboring a DNA coding for a BT-IgSF protein having a mutation. The“stringent conditions” referred to herein includes a condition underwhich a so-called specific hybrid is formed, and a non-specific hybridis not formed. For example, the stringent conditions include a conditionunder which two of DNA's having homology of not less than 20%,preferably not less than 50%, more preferably not less than 80%, arehybridized with each other, and two of DNA's having homology lower thanthe above are not hybridized with each other. Specifically, thestringent conditions are exemplified by a condition under which two ofDNA's are hybridized with each other at a salt concentrationcorresponding to 0.2×SSC, 0.1% SDS at 42° C., preferably 0.1×SSC, 0.1%SDS at 68° C.

A method for preparing a BT-IgSF protein will be explained hereafter. Alarge amount of a BT-IgSF protein can be prepared by, for example,inserting total DNA or a part of the DNA into an appropriate expressionvector having a promoter sequence, marker gene, replication origin andso forth in a conventional manner and introducing the vector into amicroorganism, cultured cell or animal or plant cell to express thevector. A variety of such expression vectors are commercially availableand can be used for the present invention. When expression of a maturedBT-IgSF protein is intended, a DNA fragment coding for the amino acidsequence of the amino acid numbers 1 to 409 of SEQ ID NO: 2 in which aninitiation codon (ATG) is added to the 5′ end, a termination codon isadded to the 3′ end and restriction enzyme recognition sequences areadded outside these codons can be inserted into the vector downstreamfrom the promoter sequence. To add these codons and restriction enzymerecognition sequences to the DNA of the present invention,oligonucleotide primers used for amplification of the DNA by PCR can bedesigned to contain the aforementioned codons and restriction enzymerecognition sequences. When the expressed BT-IgSF protein is accumulatedin a microbial cell as inclusion bodies, they can be solubilized with a8 M urea solution, 6 M guanidine hydrochloride solution or the like,purified by column chromatography and refolded to produce a BT-IgSFprotein having a structure the same as or similar to a naturallyoccurring protein.

Further, a BT-IgSF protein can be expressed by, for example, inserting aDNA coding for the BT-IgSF protein into a plasmid having an adenoviruspromoter, DHFR (dehydrofolic acid reductase) gene, SV40 poly-A sequenceand replication origin by ligation, introducing the obtained plasmidinto a DHFR-deficient CHO cell (cell derived from Chinese hamster) by acalcium phosphate method or the like and culturing the cell in MTX(methotrexate) medium. In this case, although a matured BT-IgSF proteinmay be directly expressed, a precursor protein may be expressed byintroducing a DNA coding for the BT-IgSF precursor having a signalsequence into the cell. When the expressed protein is accumulated as asoluble protein in a cell culture medium, the BT-IgSF protein can beobtained by concentrating the medium using an ultrafiltration membraneand purifying the protein by column chromatography in a conventionalmanner.

The function as a cell adhesion molecule of BT-IgSF protein prepared byrecombinant DNA techniques and purified by column chromatography or thelike can be confirmed by the cell aggregation assay (K. Hirata, et al.,Cloning of an immunoglobulin family adhesion molecule selectivelyexpressed by endothelial cells, Journal of Biological Chemistry, 276,16223–16231, 2001) or the like.

As shown in the examples described later, it has been demonstrated thatBT-IgSF proteins are expressed in the testis and the brain, inparticular, in the corpus callosum in the brain most strongly.Therefore, a BT-IgSF protein is expected to function as a cell adhesionmolecule involved in the development of the corpus callosum based on itsextremely unique characteristic that its expression is limited in thecorpus callosum in the brain. Further, the position of the BT-IgSF geneon the human chromosome was searched in the public human genomedatabase. As a result, it was found that the BT-IgSF gene located on thehuman chromosome 3 (3q12-23).

The sequence corresponding to the BT-IgSF gene in the database wasinterrupted by at least 5 introns, and the sequences of the codingregion and exons were unknown. However, since the coding sequence of theBT-IgSF gene has been elucidated by the present invention, thefunctional structure of the genomic gene has also been elucidated.

Based on the above, reports about cases where aplasia of the corpuscallosum was considered to be caused by deletion of a region on thechromosome 3 in which presence of the BT-IgSF gene was elucidated weresearched for. As a result, it was found that three of such cases hadbeen reported to date (Genuardi, M., Calvieri, F., Tozzi, C., Coslovi,R., Neri, G., A new case of interstitial deletion of chromosome 3q,del(3q)(q13.12q21.3), with aplasia of the corpus callosum, ClinicalDysmorphology, 3, 292–296, 1994; Ogilvie, C. M., Rooney, S. C., Hodgson,S. V., Berry, A. C., Deletion of chromosome 3q proximal region gives toa variable phenotype, Clinical Genetics, 53, 220–222, 1998). These casesshowed predominant symptoms of facial malformation, mental retardation,speech disorder and so forth due to aplasia of the corpus callosum,which were considered to be caused by deletion of the chromosome 3.However, the causative gene is still unidentified and hence no treatmentmethod has been established. Since the BT-IgSF gene identified by thepresent invention is a gene that exists in the aforementioned deletedregion and prominently expressed in the corpus callosum, and moreover itis a cell adhesion molecule, which is considered to be important fordevelopment of the corpus callosum, this gene is very likely to be acausative gene of these cases.

Therefore, the BT-IgSF gene is considered to be useful for diagnosis ofcorpus callosum aplasia cases. For example, a disease associated withaplasia of the corpus callosum can be diagnosed by investigatingexistence or expression of the BT-IgSF gene by using PCR primers orhybridization probe containing the nucleotide sequence of SEQ ID NO: 1or a partial sequence thereof. Specifically, the existence or structureof the BT-IgSF gene can be confirmed by PCR using chromosomal DNA of asubject as a template and the aforementioned primers or hybridizationusing the chromosomal DNA and the aforementioned probe. Further,expression of the BT-IgSF gene can be examined by performing PCR orhybridization using mRNA extracted from an appropriate tissue. When theBT-IgSF gene is deficient or is not expressed even though it is notdeficient, onset of the diseases is suspected based on such a fact.

As the aforementioned primers, for example, oligonucleotides having thenucleotide sequences of SEQ ID NOS: 8 and 9 can be mentioned. Inaddition to these primers, primers can be prepared from an arbitrarypart in the sequence of SEQ ID NO: 1. Further, the BT-IgSF gene may alsobe useful as an agent for gene therapy containing this gene or a vectorincorporated with this gene in treatment of the aforementioned cases.Further, the BT-IgSF protein itself can be used for treatment of aplasiaof the corpus callosum or the like.

Further, as the aforementioned hybridization method, for example, the insitu hybridization method can be specifically mentioned. The probe canbe prepared by inserting cDNA of the BT-IgSF gene into a vector such aspCR2.1 vector (Invitrogen) and performing in vitro RNA transcription ina conventional manner. In this case, if a labeled probe is prepared byusing digoxigenin or the like, a tissue section can be stained by usingsuch a probe (Murase, S., Hayashi, Y., Expression pattern andneurotropic role of the c-fms proto-oncogene M-CSF receptor in rodentpurkinje cells, The Journal of Neuroscience, 24, 10484–10492, 1998).

Further, since the BT-IgSF gene has a characteristic that its expressionis limited to the testis apart from some tissues in the brain asmentioned above, it is expected to function as a cell adhesion moleculeinvolved in adhesion between cells in the testis. The seminiferoustubule of the testis is a place of spermatogenesis, where aspermatogonium differentiates into a spermatocyte, resulting inspermatogenesis. While hormones are important in this sperm maturationprocess, it is widely known that nursing cells also play an importantrole. The nursing cell is considered to function by directly adhering toa spermatogonium or spermatocyte and is expected to require a celladhesion molecule for this adhesion. In fact, it has been reported thatsome cell adhesion molecules are expressed in the testis. For example,N-cadherin is a well-known cell adhesion molecule and it has beenreported that it is also expressed in the testis. However, its role inspermatogenesis has not been elucidated so far (Cyr, D. G., Blaschuk, O.W., Robaire, B., Identification and developmental regulation of cadherinmessenger ribonucleic acids in the rat testis, Endocrinology, 131,139–145, 1992).

Since a BT-IgSF protein is obviously a cell adhesion molecule based onits structure and is specifically expressed in the testis, this proteinis strongly expected to contribute to, for example, adhesion betweennursing cells and spermatogoniums or between nursing cells andspermatocytes and play an important role in spermatogenesis. Therefore,the BT-IgSF gene is considered to be also useful for diagnosis andtreatment of aspermatogenesis.

Expression of the BT-IgSF gene in the testis, in particular, in theseminiferous tubule, can be confirmed in the same manner as in theconfirmation of the expression of the BT-IgSF gene in the corpuscallosum described above.

Further, an agent for gene therapy containing the BT-IgSF gene or avector incorporated with this gene or the BT-IgSF protein itself can beused in treatment of aspermatogenesis or the like.

Furthermore, since the amino acid sequence of BT-IgSF protein and thenucleotide sequence of the gene coding for the same have been elucidatedby the present invention, BT-IgSF gene homologues in other animals canbe easily obtained based on this information. A model animal deficientin the BT-IgSF gene can be prepared by using the BT-IgSF gene of thepresent invention or its homologue.

EXAMPLES

The present invention will be explained more specifically with referenceto the following examples.

<1> Search and Isolation of Gene Coding for Amino Acid Sequence HavingHomology to Amino Acid Sequence of Extracellular Region of M-CSFReceptor

Based on the amino acid sequence of the extracellular region of theM-CSF receptor, an expressed sequence tag (EST) clone coding for anamino acid sequence having homology to the sequence was searched in adatabase. As the database, the mouse EST database of the National Centerfor Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) wasused, and tblastn was used as the homology search program (see Altschul,S. F., Madden, T. L., Scaffer, A. A., Zhang, J., Miller, W., Lipman, D.J., Gapped BLAST and PSI-BLAST: A new generation of protein databasesearch programs, Nucleic Acids Research, 25 (17), 3389–3402, 1997).

As a result of the search, one mouse EST clone (#BF236252) was obtained.In the EST database, nucleotide sequences of cDNAs randomly extractedare roughly determined are registered, and the nucleotide sequences ofindividual cDNAs lack accuracy. Further, the registered sequences do notreflect complete mRNA sequences, but only parts thereof are registered.Therefore, the gene fragment was accurately sequenced based on thesequence of #BF236252. Specifically, the sequencing was performed asfollows.

Based on the nucleotide sequence of the aforementioned EST clone(#BF236252), oligonucleotides having the nucleotide sequences of SEQ IDNOS: 3 and 4 were prepared. Then, a cDNA fragment corresponding to theEST clone was amplified by PCR using the oligonucleotides as primers andcDNA derived from the mouse testis (Clontech, Catalog No. K1429-1) as atemplate. PCR was carried out by using a commercially available kit(Clontech, Advantage 2 PCR Kit (#K1910)) and a PCR apparatus (TakaraShuzo, PCR Thermal Cycler MP) according to the attached protocol in aconventional manner.

The obtained PCR-amplified fragment was inserted into the pCR2.1 vectorby using an Original TA Cloning Kit (Invitrogen, #K2000) according tothe attached protocol and cloned in an Escherichia coil strainINValphaF′. The plasmid DNA was prepared by using Plasmid Mini Kit(QIAGEN) according to the manufacturer's protocol. The sequence of theobtained plasmid DNA was determined by performing a reaction usingBigDye Terminator Cycle Sequencing FS Ready Reaction Kit (AppliedBiosystems) and M13 Reverse Primer and the M13 Forward (−20) Primeraccording to the attached protocol and then performing electrophoresisby using 373S DNA Sequencer (Applied Biosystems).

Subsequently, 3′ rapid amplification of cDNA ends (3′ RACE) was carriedout to obtain a cDNA region (3′ end side) that was not included in theclone obtained as described above and the aforementioned EST clone.First, a primer having the nucleotide sequence of SEQ ID NO: 5 wasprepared based on the sequence of the above-obtained cDNA fragment.Then, 3′-RACE-Ready cDNA was synthesized by using mRNA derived from themouse testis (Clontech, #6612-1) as a template and SMART RACE cDNAAmplification Kit (Clontech, #K1811) according to the attached protocol.PCR amplification was performed by using the obtained cDNA, theaforementioned primers and PCR Thermal Cycler MP (Takara Shuzo)according to the protocol attached to the kit to obtain a cDNA fragmenton the 3′ end side of the target gene. The amplified fragment was clonedto obtain plasmid DNA by the aforementioned method, and its nucleotidesequence was determined. Thus, the cDNA sequence of the target genehaving a substantially full length was determined except for a part onthe 5′ end side.

Subsequently, to identify a human homologue of the gene, the human ESTdatabase was searched based on an amino acid sequence estimated to beencoded by the above obtained mouse cDNA in the same manner as describedabove. As a result, one human EST clone (#AA620978) was obtained. Asdescribed above, it was considered that the sequence of this EST clonewas not accurate either and did not include the coding region in itsfull length. Therefore, 3′ RACE and 5′ RACE were performed to obtain thetarget cDNA in the full length.

A primer for 3′ RACE (SEQ ID NO: 6) and a primer for 5′ RACE (SEQ ID NO:7) were prepared. Then, 3′-RACE-Ready cDNA and 5′-RACE-Ready cDNA weresynthesized by using mRNA derived from the human kidney (Clontech,#6538-1) as a template and SMART RACE cDNA Amplification Kit (Clontech,#K1811) according to the attached protocol. These cDNA were amplified byPCR using the aforementioned primers and PCR Thermal Cycler MP (TakaraShuzo) according to the protocol attached to the kit to obtain cDNAfragments on the 3′ end side and 5′ end side of the target gene. Theamplified fragment was cloned to obtain plasmid DNA by theaforementioned method, and its nucleotide sequence was determined. Thus,the complete nucleotide sequence of the target gene and the amino acidsequence encoded by the gene were determined. These nucleotide sequenceand amino acid sequence are shown in SEQ ID NOS: 1 and 2, respectively.

The amino acid sequence was searched by using the Simple ModularArchitecture Research Tool (SMART, see Schulta, J., Copley, R. R.,Doerks, T., Ponting, C. P., Bork, P., SMART: A web-based tool for thestudy of genetically mobile domains, Nucleic Acids Research, 28 (1),231–234, 2000). As a result, it was found that the protein encoded bythe aforementioned cDNA was constituted by a signal peptide,extracellular region, transmembrane region and intracellular region fromthe N-terminus side. Further, it was also found that the extracellularregion was constituted by two immunoglobulin-like regions. Therefore,this protein is a protein molecule that exists on the cell surface andbelongs to the immunoglobulin superfamily. This protein was designatedas BT-IgSF.

Known proteins that show homology to the amino acid sequence of theBT-IgSF protein were searched by the aforementioned method(http://www.ncbi.nlm.nih.gov/BLAST/). As a result, as protein moleculeshaving the highest homology at the amino acid level, there were found anendothelial cell-selective adhesion molecule (see Hirata, K., Ishida,T., Penta, K., Rezaee, M., Yang, E., Wohlgemuth, J., Quertermous, T.,Cloning of an immunoglobulin family adhesion molecule selectivelyexpressed by endothelial cells, Journal of Biological Chemistry, 276(19), 16223–16231, 2001) and a receptor for coxsackie virus andadenovirus (see Bergelson, J. M., Cunningham, J. A., Droguett, G.,Kurt-Jones, E. A., Krithivas, A., Hong, J. S., Horwitz, M. S., Crowell,R. L., Finberg, R. W., Isolation of a common receptor for coxsackie Bviruses and adenoviruses 2 and 5, Science, 275, 1320–1323, 1997).However, both molecules showed homology of only about 30% to BT-IgSF atthe amino acid level. Further, in the search at the nucleotide sequencelevel, no molecule showing significant homology was found (10% orlower).

<2> Analysis of BT-IgSF Gene Expression

In order to find the function of the BT-IgSF molecule, tissues in whichthe molecule was expressed were examined by using Human Multiple TissueExpression Array (Clontech, #7775-1) by the Northern hybridizationmethod. The hybridization was performed according to the protocolattached to the array, and the procedure is outlined below.

First, a cDNA probe including the entire coding region of cDNA of thehuman BT-IgSF gene was prepared. To this end, primers shown as SEQ IDNOS: 8 and 9 were prepared. A cDNA fragment derived from the humanBT-IgSF gene was amplified by PCR using these primers and cDNA derivedfrom the human kidney (Clontech, Catalog No. K1420-1) as a template. PCRwas carried out by using Advantage 2 PCR Kit (Clontech, K1910) and PCRThermal Cycler MP (Takara Shuzo) in a conventional manner according tothe attached protocol. The obtained cDNA has one base of G at the 5′ endand a sequence of GACATGAGG at the 3′ end in addition to the sequence ofSEQ ID NO: 1. This cDNA was labeled by using ³²P-labeled dCTP (AmershamPharmacia Biotech, #PB10205) and MegaPrime DNA Labeling System (AmershamPharmacia Biotech, #RPN1606) according to the manufacturer's protocol.Northern hybridization was carried out by using this labeled probe andHuman Multiple Tissue Expression Array. The hybridization was performedovernight at 65° C. by using ExpressHyb Hybridization Solution(Clontech, #8015). Washing was performed four times with 2×SSC solutioncontaining 1% SDS 65° C. for 20 minutes and twice with 0.1×SSC solutioncontaining 0.5% SDS at 55° C. for 20 minutes.

As a result of the above experiment, it was found that expression of theBT-IgSF gene was substantially limited in the testis and the brain (FIG.1). Expressions at various sites in the brain were further analyzed byusing the aforementioned array, and it was found that the BT-IgSF genewas highly expressed especially in the corpus callosum (FIG. 1).Therefore, since the BT-IgSF protein has an extremely uniquecharacteristic that its expression is limited in the corpus callosum andthe testis, this protein is expected to function as a cell adhesionmolecule involved in development of the corpus callosum andspermatogenesis.

1. A method for diagnosing increased risk of aplasia of the corpuscallosum in humans, comprising the steps of: investigating the presenceof a target gene corresponding to the nucleotide sequence of SEQ ID NO:1 by PCR using primers consisting of oligonucleotides by which thetarget gene is amplified, and diagnosing increased risk of aplasia ofthe corpus callosum when the presence of the target gene is notdetected.
 2. The method according to claim 1, wherein theoligonucleotides consist of an oligonucleotide having the nucleotidesequence of SEQ ID NO: 8 and an oligonucleotide having the nucleotidesequence of SEQ ID NO: 9.